Sandbox 11

Please do NOT make changes to this sandbox. Sandboxes 10-30 are currently reserved by Prof. Sheila Jaswal at Amherst College.

Note to the reader: This is a really boring sandbox. I left all of my toys at home. Sorry.

Nocardiopsis alba Protease A
Nocardiopsis alba Protease A, or NAPase, is an acid-resistant homolog of α-lytic protease (αLP). As such, NAPase and αLP are both kinetically stable proteases, meaning it is the large barrier to unfolding that keeps these proteases in their folded, active state. This is different from most other proteins, which stay in their folded, or native, state because of the energy difference between their native and unfolded states, with the native state being lower in energy. These proteases gain a significant advantage in half-life to unfolding because of their kinetic stability, but the advantage comes with a price. The barrier to folding is large, with αlp's half life for folding around 1800 years. Luckily, these proteases have coevolved a pro region that can assist with folding while covalently attached, or while in solution with the unfolded protease. Once the protease has been guided to its native state by the pro region, it mercilessly proteolyzes the pro region that helped it gain its protein-degrading ability.

The NAPase molecule provided shows two NAPase molecules that are mirror images, so here is just one. NAPase, along with the rest of the trypsin family, has an active site that consists of the "catalytic triad." This catalytic triad is made up of three amino acid residues (H57, D102, and S195) that play a major role in binding the substrate and catalyzing proteolysis. The distance between these residues on the protein chain, and the complexity of folding one might imagine is occurring, help to demonstrate the value of the Pro region.

One of the major features of NAPase is that each protease has two domains, an N domain and a C domain. In this N to C Rainbow, one can see the N domain (red, orange, yellow), so-called because it contains the N-terminal amino acid, is connected covalently through the protein to the C domain (green, blue, violet). The horizontal axis of this scene is the main dividing line between the domains, with few chains crossing the barrier. It is important to note here that research suggests the first step to unfolding is when the two domains split. Just ask Professor Jaswal.

Acid Resistance
Kelch (2007), looks at the differences between NAPase and αLP to try to understand what causes NAPase to be more acid resistant than αLP. It is found that they form a similar number of salt-bridges (7 in NAPase, 8 in αLP), but the salt bridges are in different places. Two of these bridges are conserved between the two, so there are five salt bridges that could be considered as important for acid resistance in NAPase. The important difference between the location of bridges is that αLP has three bridges that span the N and C domains, while NAPase has none that span the domains. The distance between the residues of each individual salt bridge in NAPase is relatively small(avg. distance = 20 residues), when compared to the large distance between the residues in αLP (avg. distance = 78 residues). This picture shows the charged residues of Glutamate and Arginine, with red representing a negative charge, and blue representing a positive charge.

So, at low pH, the domain bridging salt bridges of αlp break or weaken enough that the N and C domains split apart enough for αLP to be protealyzed. NAPase avoids this with its alternate placement of salt bridges. Of course, there may be more reasons for the added acid resistance, but those are as yet undetermined. And to make this page more fun, here is another salt bridge in NAPase. This one is the longest salt bridge in NAPase (57 residues), but it is between two residues that are both in the C domain. More importantly, if this bridge were to break, the nearby antiparallel β sheets and cysteine bridge would help to maintain the native state of NAPase.